Electrochemical plasma activated aqueous chemo therapeutics

ABSTRACT

Methods for the generation of electrochemical plasma activated aqueous chemotherapeutics (EPAAC) solutions are described. These solutions have been found to selectively reduce the proliferation of human pancreatic cancer cells, with no toxic effects for healthy cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. patentapplication Ser. No. 15/585,084 for “Electrochemical Plasma ActivatedAqueous Chemotherapeutics”, by Brooks M. Hybertson et al., which wasfiled on 2 May 2017 and claims the benefit of U.S. Provisional PatentApplication No. 62/330,713 for “Methods and Applications ofElectrochemical Plasma Activated Aqueous Chemotherapeutics” by Brooks M.Hybertson et al., which was filed on 2 May 2016, the entire contents ofwhich patent applications are hereby specifically incorporated byreference herein for all that they disclose and teach.

BACKGROUND

In recent years, the physicochemical characteristics of non-thermalplasmas have been extensively studied in the field of Plasma Medicine,including preliminary investigations of possible use to treat biologicaldisorders such as infections, wounds, and cancer. Plasma refers to anionized, electronically-excited gas that contains ions, radicals, andelectrons. The term non-thermal indicates a plasma comprised of gasatoms, molecules, and ions in which their kinetic energy is low enoughthat the overall fluid temperature remains close to, or at, roomtemperature while the electrons encompassed in the plasma contain higheramounts of energy. This additional kinetic energy allows the electronsto induce the cleavage of chemical bonds, but the low overall kineticenergy of the plasma can suppress the complete atomization of themolecule.

Non-thermal plasma species identified for interaction with biologicalsystems including cancer cells include hydrogen peroxide, ozone, nitriteanion, and nitrate anion, with additional species such as peroxynitriteanion, other NON, free radical, and short- or long-lived reactiveintermediates being generated. Notably, the combination of plasmaspecies appears to be more potent than individual chemical components.The selectivity of non-thermal plasma species at inhibiting cancercells, but not normal cells, in prior work, demonstrates promise for thefield of Plasma Medicine relating to cancer treatments.

SUMMARY

To achieve the purposes of embodiments of the present invention, asembodied and broadly described herein, the method for treatingpancreatic cancer hereof, includes: providing a solution having a chosenquantity of sodium chloride dissolved in water; providing an apparatuscomprising: a chamber for containing the solution, the chamber having anelectrically conducting cylindrical wall having an axis, an inlet forthe solution at one end thereof and an outlet for the solution at theopposing end thereof; an elongated hollow, electrically conducting shaftrotatably disposed collinearly with the axis of the cylindrical portionof the chamber and having an open end and a closed end, the closed endthereof facing the end of the chamber having the inlet for the solution;at least one electrically conducting pin electrode having a boretherethrough, a first end and a second end, and having a frit effectivefor generating bubbles in the solution at the surface of the at leastone pin electrode from which a plasma discharge is generated, disposedin the bore in the vicinity of the first end thereof, the second endbeing mounted through the surface of the shaft such that the bore of theat least one pin electrode is in gaseous communication with the interiorof the hollow shaft; an electrically insulating cylinder having an axiscollinear with the axis of the chamber disposed within the chamber, theelectrically insulating cylinder having a surface through which aportion of the at least one pin electrode extends, the electricallyinsulating cylinder rotating with the shaft; a motor for rotating theshaft; a solution inlet for introducing the solution into the inlet ofthe chamber such that the solution flows axially in the cylindricalportion of the chamber; a gas source for introducing a chosen gas intothe open end of the shaft at a pressure such that gas bubbles exitthrough the frit of the at least one pin electrode and rise in acounter-current manner to the flow of the solution; and an electricalpower supply for applying a voltage to the shaft effective forinitiating and maintaining the plasma discharge between the first end ofthe at least one pin electrode and the interior of the conductingcylindrical portion of the chamber; introducing the solution into thechamber through the solution inlet; introducing the chosen gas from thegas source into the open end of the shaft; rotating the shaft at achosen rotational rate using the motor; applying a selected voltage fromthe power supply to the shaft, whereby a plasma discharge is generated;exposing the solution to the plasma for a selected period of time,forming thereby a plasma activated solution having a pH; and exposingcells afflicted with pancreatic cancer to the plasma activated solution.

In another aspect of embodiments of the present invention and inaccordance with its purposes the method for treating pancreatic cancerhereof, includes: providing a solution having a chosen quantity of asingle sodium chloride solute dissolved in water; exposing the singlesodium chloride solute solution to an atmospheric pressure plasma for aselected period of time, forming thereby a plasma activated solutionhaving a pH; adding a base to the plasma activated solution to adjustthe pH to 7.4±0.1, forming thereby a physiological solution; addingsodium chloride to the physiological solution to adjust the chosenquantity of sodium chloride to 9±0.1 g per liter of solution, formingthereby an isotonic solution; and exposing cells afflicted withpancreatic cancer to the isotonic solution.

Benefits and advantages of embodiments of the present invention include,but are not limited to, providing a method for generatingelectrochemical plasma activated solutions which are selective inreducing the proliferation of pancreatic cancer cells, while exhibitingminimal cytotoxic effects on healthy cells, such as smooth muscle cells,endothelial cells, and other organ-specific cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and form a part ofthe specification, illustrate the embodiments of the present inventionand, together with the description, serve to explain the principles ofthe invention. In the drawings:

FIG. 1A is a schematic representation of a perspective view of anembodiment of the tubular high-density plasma reactor (THDPR) used togenerate the activated aqueous chemotherapeutic solutions hereof,illustrating the fluid and gas flow and a pin electrode configuration,FIG. 1B is a schematic representation of the side view of an unmodifiedpin electrode, and FIG. 1C is a schematic representation of the sideview of a pin electrode in which a frit is inserted into the borethereof.

FIG. 2 shows graphs of human pancreatic cell proliferation for salinecontrol solution, carboplatin positive control drug, and 5 min.electrochemical plasma activated aqueous chemotherapeutics (EPAAC)solution, referred to here as aqueous plasma chemotherapeutics (APC),where the following dosages were administered to the cancer cellsdiluted in appropriate complete growth medium for each cell type: (1) 1v/v % for saline and 5 min. APC, 0.2 μg/mL for carboplatin; (2) 5 v/v %for saline and 5 min APC, 1 μg/mL for carboplatin; (3) 12.5 v/v % forsaline and 5 min. APC, 2.5 μg/mL for carboplatin; (4) 25 v/v % forsaline and 5 min. APC, 5 μg/mL for carboplatin; and (5) 50 v/v % forsaline and 5 min. APC, 10 μg/mL for carboplatin.

FIG. 3 shows graphs of human pancreatic cancer cell proliferation as afunction of dosage for 5 min. and 10 min. APC, for the followingdosages: 1, 5, 12.5, 25, and 50 v/v %, illustrating that dose-dependentcell proliferation inhibition occurs for the 5 min. APC solution,whereas the 10 min. APC does not yield a dose-dependent cellproliferation inhibition.

FIG. 4 shows graphs of cell proliferation for several human cell speciesas a function of APC solution dosage for the following dosages: 1, 5,12.5, 25, and 50 v/v %, illustrating the non-toxic nature of APCsolutions when administered to a variety of normal human cell lines.

FIG. 5 shows graphs of human pancreatic cancer cell proliferation forAPC solution and a “mock” APC solution, for the following dosages: 1, 5,12.5, 25, and 50 v/v %, illustrating that the “mock” APC solution doesnot yield the same dose-dependent pancreatic cancer cell growthinhibition as does the APC solution.

FIG. 6 is a graph of the oxidant level as a function of residence timein the plasma, illustrating that the oxidant level increases as afunction of increasing residence time in the reactor.

FIG. 7 is a graph of the measured ORP as a function of time for aspecific example of a 300 mg/L TDS influent solution where the power wasturned on and off to yield periods of plasma discharge and no currentflowing, respectively, illustrating that the measured ORP of thesolution increases when the plasma is on, and decreases when the plasmais off.

FIG. 8 is a graph of the oxidation of Yellow 5 dye as a function ofapplied current.

FIG. 9 is a graph of the oxidation of Yellow 5 dye as a function ofapplied power.

FIG. 10 is a graph of plasma quality as a function of total dissolvedsolids in solution for several electrode/cylinder wall spacings for theplasma generator, illustrating that the plasma quality, a qualitativescore based on visualization of plasma discharge flashes through atransparent window into the THDPR, as well as data collection indicatingsteady power, voltage, and current parameters, is dependent on the totaldissolved solids (TDS) of the influent solution, as well as on theelectrode gap.

DETAILED DESCRIPTION

In accordance with embodiments of the present invention, activatedspecies generated in aqueous solutions of sodium chloride in a tubular,high-density plasma reactor, described in detail below, and termed aselectrochemical plasma activated aqueous chemotherapeutics (EPAAC), oraqueous plasma chemotherapeutics (APC), have been used for treatment ofpancreatic cancer. The EPAAC solution may be directly administered tothe pancreas for a localized effect by use of a catheter or syringe orby intravenous administration. The EPAAC solution is useful as a cancertherapy due to its ability to selectively inhibit the proliferation ofhuman pancreatic cancer cells, while exhibiting non-toxic behaviortoward normal human cells. Such solutions have been found to selectivelyand efficiently inhibit cancer cell proliferation on the same order, oreven more efficiently, than established chemotherapeutic drugs whilemitigating cytotoxic effects. Notably, common small-moleculechemotherapy drugs are not selective in nature, thus, normal cells aretargeted alongside cancer cells, resulting in undesirable and damagingside effects. The selectivity and mechanism of action is expected to bedependent upon solution composition and electrochemical treatmentparameters. Treatment of pancreatic cancer may employ EPAAC alone, or incombination with chemotherapeutic or other additives to result inenhanced cellular uptake of drugs for improved oncological performance.

Reference will now be made in detail to the present embodiments of theinvention, examples of which are illustrated in the accompanyingdrawings. In the FIGURES, similar structure will be identified usingidentical reference characters. It will be understood that the FIGURESare presented for the purpose of describing particular embodiments ofthe invention and are not intended to limit the invention thereto.

A. Apparatus:

Turning first to FIG. 1 , a schematic representation of a perspectiveview of an embodiment of the tubular high-density plasma reactor, 10,used to prepare EPAAC solutions in accordance with embodiments of thepresent invention is shown, illustrating sodium chloride solution, 12,entering tubular chamber or container, 14, through inlet, 16, in upperflange, 18, exiting as a plasma activated solution, 20 a, and, 20 b,through outlets, 22 a, and, 22 b, respectively, in lower flange, 24,thereof, after traveling axially, 26, through tube 14. House air, 28, isintroduced into solution 26 through hollow, rotatable, electricallyconducting shaft, 30. A flow rate of about 200 SCFH was employed in theEXAMPLES. Shaft 30 is rotatably driven by motor, 32, and supports achosen configuration of hollow pin discharge electrodes, 34, affixedthereto and in communication with air 36 from air source 28. Airexiting, 36, from hollow pins 34 rises, 38, flowing counter-currently tosolution 26, and exits container 14 through orifice, 40, in upper flange18, which may include a valve. The solution travels the axial length ofreactor 10 and plasma activated solution exits through liquid orifices22 a and 22 b which may include release valves at the bottom of thereactor. As will be discussed below, the air introduction system isdesigned such that only a radial pressure gradient exists between theinner shaft and outer cylinder, thereby ensuring an equal volumetricflow rate of the air through the bores of pin electrodes 34, independentof their position along shaft 30.

As illustrated in FIG. 1A, hollow pin electrodes 34 protrude outwardfrom shaft 30, through insulating ceramic, 42, which rotates with shaft30, toward the outer stationary cylinder, 44, of container 14, and maybe arranged such that approximately 100 discharge electrodes aredisposed on a one foot length of shaft 30. Hollow pin electrodes 34 maybe fabricated using stainless steel, tungsten, titanium, or molybdenum,as examples, although stainless steel electrodes were employed in thegeneration of the EPAAC solutions in the EXAMPLES. Motor 32 is effectivefor rotating shaft 30 at a rate of between 60 rpm and 2000 rpm, onbearings, 46 a, and, 46 b. About 1000 rpm was employed in preparing theEPAAC solutions. Gaps, 48, between the tips of discharge electrodes 34and outer cylinder 44 can be adjusted to distances on the order of about0.5 mm to approximately 2 mm. An approximately 0.75 mm gap was used inthe EXAMPLES.

A plasma discharge is initiated at the outer tip of discharge electrodes34 and propagates to the inner wall of stationary outer cylinder 44. Anelectrical power supply, 50, capable of supplying between 0.5 kV and 1kV is effective for initiating and maintaining this discharge, and isplaced in electrical connection with conducting shaft 30 using carbonbrush, 52, as an example, for which the return connection to completethe circuit is in contact with stationary outer cylinder 44. Typicalvoltages used to prepare the EPAAC were between about 250 V and about750 V. Between approximately 0.5 A and about 3 A of current wereapplied. During exposure, fluid temperature was found to increasebetween about 0° C. and about 30° C. To date only DC voltages have beenused; however, AC or pulsed operating modes may also be employed.Bearings 46 a and 46 b are insulated to ensure that neither the innershaft nor the outer stationary cylinder becomes charged. Tubular plasmareactor 10 is expected to maximize the time in which a solution elementmoving axially through the reactor is in contact with the plasma. Thisis accomplished by minimizing the distance that the pin electrodesprotrude from insulating ceramic portion 42 of the rotating shaft 30.

FIG. 1B is a schematic representation of the side view of unmodified pinelectrode, 54, having bore, 56, and tapered tip, 58, disposed at thedischarge end of pin electrode 54. FIG. 1C is a schematic representationof the side view of pin electrode 54 in which frit, 60, is inserted intobore 56 in the vicinity of the discharge end, 62, thereof. Frit 60 maybe a commercially available metal or glass frit having appropriatestructural integrity. Equal volumetric flow rates of the air through thebores of pin electrodes 34, independent of their position along shaft30, may be obtained by restricting the air flow using appropriate frits.

Solution was circulated at about 2.6 gph (gallon per hour) through areactor volume of approximately 225 mL to achieve a given residencetime; additional combinations of flow rates and liquid volumes may beemployed to achieve a selected fluid plasma-exposure residence time.

B. Influent:

The influent solution can be prepared with variable quality of water,ranging from sterilized water for injection (WFI) to nanopure,deionized, or tap water, since the inherent sterilization processes thatoccur during plasma treatment impart a sterile nature to the water thatis treated. Total dissolved solids (TDS) concentration of the initialinfluent water may range from 0 to greater than 15,000 mg/L, or moreadvantageously between 1,000-7,000 mg/L. As stated above, the initialsolution contained sodium chloride in the absence of other solutes.Initial chloride ion levels can impact the intermediate chemicalspecies, both short- and long-lived, that form during the treatmentperiod, since the presence of chloride is known to accelerate ozonedecomposition in aqueous solution. Other additives may include, but arenot limited, to salts, sugars, vitamins, alcohols, ketones, acids,buffers, and drugs. Solution pH may have an effect in controlling theresulting reactive species that form. The EXAMPLES describe an initialinfluent composition comprised of 1000 mg/L USP-grade sodium chloride(NaCl), in the absence of other solutes, prepared in sterile water forinjection.

If chlorinated intermediates are not desired, alternate salts can beemployed to contribute to solution conductivity, but not result inchlorinated species. Other intermediate pathways may be favored with theaddition of nitrite or nitrate salts, as examples. Additionally, theconcentration of ions in solution may alter the fraction of appliedcurrent contributes to conductive pathways as opposed to plasmapathways. As an example, lower saline content will lower solutionconductivity, thus making plasma discharge the dominant current pathway.

C. Effluent Post-Processing:

The effluent solution may be modified post-processing to adjust thefinal properties of the EPAAC solution prior to administration as atherapeutic agent. As an example, for subsequent biological application,the TDS of the final product can be adjusted to approximately theisotonic range (9 g/L saline) by addition of sodium chloride.Additionally, the pH of the solution can be adjusted to be about thephysiological pH value 7.4. Other processing steps may includecentrifugation or filtration.

Therapeutic agents, such as chemotherapeutics and other drugs, can beadded to the EPAAC solution for increased efficacy of the desiredtherapeutic outcome. The recovered EPAAC solution can be modified byaddition of salts, sugars, vitamins, buffers, proteins, enzymes anddrugs or drug pre-cursors.

D. EPAAC Solutions for Cancer Therapy:

EPAAC solutions have demonstrated cancer cell growth inhibitionselectivity. The EPAAC solution can be administered as a direct(stand-alone) therapeutic, or in combination with other components. Inaccordance with their stability, the solutions may be stored in bags anddistributed for later clinical use. The shelf lives of the EPAACsolutions investigated have been found to be on the order of days orweeks.

Reactor systems may be delivered to treatment sites for on-sitegeneration of EPAAC solution for patient administration. EPAAC solutionsmay be injected subcutaneously directly at the site of a tumor forlocalized treatment, or intravenously for systemic treatment. Otherroutes of administration include use of catheters.

The EPAAC solutions generated in accordance with the teachings ofembodiments of the present invention has been found to be selective inreducing the proliferation of pancreatic cancer cells, while exhibitingminimal cytotoxic effects on healthy cells, such as smooth muscle cells,endothelial cells, and other organ-specific cells.

Having generally described embodiments of the present invention, thefollowing EXAMPLES provide additional details.

a. Apparatus Parameters:

In what follows, an approximately 2 mm electrode gap, an about 200 SCFHflow of house air, an approximately 1000 rpm rotation rate (shaft 30),an about 2.6 gph flow rate through an approximately 225 mL reactorvolume, and an about 3 A steady-state DC current were used. Thesolutions were treated for about 5 min. in a recirculation mode. Thesolution can be recirculated through a single reactor to increase theconcentration of the activated aqueous species present in solution byincreasing the solution residence time in the reactor, or the effluentfrom one reactor can be introduced into a second reactor in series withthe first. The inlet gas used during the plasma generation can alsosignificantly impact the intermediates generated, such as the increasedformation of NO_(x) species (i.e. nitrite/nitrate and theircorresponding acids) with the use of air and N₂ as the inlet gas whencompared to using high-purity O₂. Further, the use of inert gases, suchas argon, for atmospheric plasma discharges has been found to yieldhydrogen peroxide-dominated solution chemistries, whereas inclusion ofoxygen yields mechanisms that favor atomic oxygen with subsequentchlorine chemistries.

b. Cell Lines and Culture:

All cells were purchased from publicly available sources, were notmodified, and were handled according to supplier specifications forcomplete growth medium, culturing, subculturing, and cryopreservationprotocols. All media formulations were obtained from publicly availablesources and prepared according to supplier specification and standardcell culture practices under sterile conditions. All live cells werereceived from the supplier under cryopreservation and cultured accordingto standard practices.

All lines used were adherent human cell lines.

-   -   (i) Pancreatic cancer cell line: MIA PaCa-2        -   Source: Available from ATCC (ATCC® CRL-1420™)        -   Complete growth medium: Eagle's Minimum Essential Medium            containing 5% fetal bovine serum and 5% cosmic calf serum    -   (ii) Human umbilical vein endothelial cells: HUVEC        -   Source: Available from ATCC (ATCC® PCS-100-010™) or Lonza            (single or pooled donor)        -   Complete growth medium: EGM-2 BulletKit (Lonza)    -   (iii) Human foreskin fibroblasts: HFF-1        -   Source: Available from ATCC (ATCC® SCRC-1041™)        -   Complete growth medium: Dulbecco's Modified Eagle's Medium            containing 15% fetal bovine serum    -   (iv) Immortalized pancreas duct epithelial-like cells:        hTERT-HPNE        -   Source: Available from ATCC (ATCC® CRL-4023™)        -   Complete growth medium: Specialty Dulbecco's Modified            Eagle's Medium composition, protocol available from ATCC    -   (v) Primary pancreatic stellate cells: Panc stellate        -   Source: Available from Applied Biological Materials (T4215)        -   Complete growth medium: Prigrow I containing 10% fetal            bovine serum.

Each cell line was adherent in a growth flask, incubated at 37° C. and5% CO₂; de-attached cell from growth flask using Trypsin-EDTA solution(0.25% Trypsin, 0.53 mM EDTA); and counted by Trypan blue stainingstandard protocol. Split cells were handled according to supplierspecification during culture.

c. Cell Proliferation Assay:

A standard cell proliferation assay was used, the Alamar blue assay, toassess quantification of viable cells. This assay is a well-establishedmethod in the scientific community and test kits and protocols arepublicly available; for example, a test kit is available from ThermoFisher Scientific. A probe compound, resazurin, was added to completegrowth medium to which the adhered cells were exposed in a standard96-well plate format. In the presence of metabolic cellular activity,resazurin is converted to resorufin, a fluorescent product. Thefluorescence measured in the solution is thus proportional to the amountof live cells present at the end of an assay period. This is a standardmethod for measuring cell proliferation.

i. Day 1: Cell Seeding:

-   -   All cell lines were collected, counted, and diluted according to        the same procedure to obtain a 10,000 cell/mL suspension of        cells in complete growth medium;    -   Each cell line was plated (seeded) in a 96-well plate layout        using commercially available Thermo Scientific™ Nunc™ MicroWell™        96-Well Microplates containing a Nunclon™ Delta cell culture        treated surface to promote cell attachment and growth. Such        plates are standard for antibiotic screens, cell-based assays,        and screening compounds; and    -   Plates were incubated for 24 h to allow cell attachment and        proliferation on the plate surface

ii. Day 2: Dosing:

-   -   For all plates, complete growth medium was removed from all        wells and replaced with fresh growth medium at volumes specific        to the dose dilution for that well (see TABLE below);    -   For APC dosing, APC was aliquoted into complete growth medium in        each well at a volume specific to the desired v/v % APC        concentration:

TABLE V_(APC) V_(media) % APC (μL) (μL) 50 100 100 25 50 150 12.5 25 1755 10 190 1 2 198 0 (Control) 0 200

-   -   For positive control, cells were independently dosed with a        carboplatin chemotherapeutic drug to ensure correct cellular        growth behavior. The dosing procedure was accomplished similarly        to that of APC; and    -   Plates were incubated for 72 h to allow impact of drug (APC or        carboplatin) on cells, followed by growth of cells to assess        ultimate impact of the drug on cell growth, according to        standard practice.

iii. Day 5: Reading

-   -   For all plates, the complete growth medium/drug solutions were        removed and the remaining adhered cells rinsed with phosphate        buffered saline to remove any residual drug that could interfere        with the fluorescent assay; cells remained adhered to the well        during this step;    -   To each well, fresh complete growth medium was added;    -   To each well, Alamar blue solution was added at 10 v/v % of the        amount of complete growth medium;    -   Plates were incubated for 1 h to allow the conversion reaction        to the fluorescent product as a result of cellular metabolic        activity to take place;    -   Plates were read using a fluorescent microtiter plate reader        according to standard procedure; the excitation wavelength        (λ_(ex)) at 530 nm and the emission wavelength (λ_(em)) at 590        nm; and    -   All fluorescent counts were normalized by the control response        (no drug, wells containing only cells and 100% complete growth        medium over the duration of the cell culture assay).

EXAMPLE 1

EXAMPLE 1 illustrates one method of producing APC solution and itssubsequent action on pancreatic cancer cells compared to a commerciallyavailable small molecule chemotherapeutic and a saline solution control.APC solution was prepared by adding 1000 mg/L USP-grade sodium chloride(NaCl) salt to 1 L of water for injection (WFI) in a sterile 1 L glasscontainer. The influent solution was treated in the tubular high-densityplasma reactor, THDPR under the following conditions: 2 mm electrodegap, introduction of 200 SCFH of house air, rotor spin rate of 1000 rpm.Once the reactor fill volume of 225 mL was achieved, continuous flow wasachieved via peristaltic pumping at 2.6 gph flow rate in a recirculationloop. Plasma discharge was initiated by applying a 3 A steady-statecurrent to the system. The solution was recirculated such that the same1 L volume cycled continuously from the reservoir to the reactor fortreatment. After a 5 min. treatment time, corresponding to an about 1min. residence time of plasma exposure, the current was stopped and theplasma-treated solution was recovered.

The recovered solution was centrifuged, the supernatant solution was pHadjusted to about 7.4 by addition of strong base (0.1 M NaOH), andbrought to isotonic by addition of USP-grade NaCl to reach 9000 mg/Ltotal dissolved solids. Within approximately 1 h of solutionpreparation, seeded MIA PaCa-2 cells adhered to the bottom of 96-wellplates were dosed with APC solution at the following concentrationsrelative to the total volume (200 μL) in the well: 1, 5, 12.5, 25, and50 v/v %, the remaining volume being composed of cell medium. Controlcells were also dosed with the isotonic saline solution treated in thesame manner as the APC, without the plasma treatment. A positive controlwas included by dosing the MIA PaCa-2 cells with carboplatin atconcentrations of 0.2, 1, 2.5, 5, and 10 μg/mL, which is an establishedtherapeutic range for carboplatin.

Cell proliferation was assessed after a 72 h incubation period, wherebythe cells were rinsed with phosphate buffered saline, PBS, given freshmedia, and subsequently reacted with Alamar blue, which was converted tothe fluorescent product, resorufin, in the presence of metabolicallyactive cells. The fluorescence counts were read using a microtiter platereader to determine the fraction of live cells compared to the controlcell wells exposed only to media.

FIG. 2 shows graphs of human pancreatic cell proliferation for saline,carboplatin, and 5 min. APC, where the following dosages wereadministered to the cancer cells: (1) 1 v/v % for saline and 5 min. APC,0.2 μg/mL for carboplatin; (2) 5 v/v % for saline and 5 min. APC, 1μg/mL for carboplatin; (3) 12.5 v/v % for saline and 5 min. APC, 2.5μg/mL for carboplatin; (4) 25 v/v % for saline and 5 min. APC, 5 μg/mLfor carboplatin; and (5) 50 v/v % for saline and 5 min. APC, 10 μg/mLfor carboplatin. It may be observed that the saline solution yielded adose-dependent proliferation of cancer cells. However, the carboplatinand APC solutions yielded dose dependent cell growth inhibition, whereat a dosage range indicated, the APC was at least as effective as thecarboplatin. These results demonstrate the dose-dependent response ofinhibition of pancreatic cancer cell growth using an APC solution. Thus,this method represents a treatment of pancreatic cancer. Efficacy ofEPAAC solutions against pancreatic cancer cells has thus beendemonstrated in the dosage range of 1-50 v/v %.

EXAMPLE 2

EXAMPLE 2 illustrates the effect of plasma residence time on thesubsequent chemotherapeutic activity of the APC solution. APC solutionwas prepared according to the description in EXAMPLE 1; however, inaddition to a 5 min. treatment time (˜1 min residence time), a 10 min.treatment time was also employed (˜2.5 min. residence time). MIA PaCa-2pancreatic cancer cells were dosed with the 5 and 10 min. APC solutions,and a proliferation assay performed. FIG. 3 are graphs of humanpancreatic cancer cell proliferation as a function of dosage for 5 min.and 10 min. APC solutions, for the following dosages: 1, 5, 12.5, 25,and 50 v/v %. It may be observed from FIG. 3 that dose-dependent cellproliferation inhibition occurs for the 5 min. APC solution, whereas the10 min. APC does not yield a dose-dependent cell proliferationinhibition, thereby indicating that the resulting chemotherapeuticactivity may be related to the plasma treatment time associated with theAPC solution preparation.

EXAMPLE 3

FIG. 4 shows graphs of cell proliferation for several human cell speciesas a function of APC solution dosage for the following dosages: 1, 5,12.5, 25, and 50 v/v %, illustrating the non-toxic nature of APCsolutions when administered to a variety of normal human cell lines. TheAPC solution generation was accomplished according to the description inEXAMPLE 1, where the APC solution was administered to adherent humannormal healthy cells in addition to human cancer cells; morespecifically, human umbilical vein endothelial cells (HUVEC), humanforeskin fibroblasts (HFF-1), immortalized pancreas duct epithelial-likecells (hTERT-HPNE), and primary pancreatic stellate cells (Panc.stellate), employed as control cell lines. It may be observed that the 5min. APC solutions yielded a dose-dependent growth inhibition of the MIAPaCa-2 cancer cells, whereas the 5 min. APC solutions failed to exertsimilar growth inhibition when applied to a variety of healthy humancontrol cell lines. More specifically, the HUVEC, HFF-1, and hTERT-HPNEcontrol lines were unaffected by APC solution treatment with 77, 88, and106% average viability, respectively, across all concentrations of APCsolutions applied. The Panc. stellate control line did exhibit somegrowth inhibition at the higher 12.5 and 25 v/v % APC concentrations,but the viable cell number was still greater than the MIA PaCa-2 cellsat those APC concentrations. These results indicate the ability of APCto selectively treat of cancer cells while remaining non-toxic tohealthy cells.

EXAMPLE 4

EXAMPLE 4 illustrates that the stable oxidant species alone are notresponsible for the therapeutic efficacy of the APC solution, and thatthe electrical discharge step is a required step. There are severalstable oxidant species that survive downstream of the electrolyticprocess, which contribute to the overall oxidant levels. Morespecifically, the following levels of oxidants have been measured: 0.5-1mg/L free chlorine, 1-10 mg/L hydrogen peroxide (H₂O₂), 0.25-1 mg/Lozone (O₃), 9 μM nitrite (NO₂ ⁻ ), and 6.5 μM nitrate (NO₃ ⁻ ). Forconsideration of whether the therapeutic effects of the APC solution canbe accounted for by adding stable oxidant reagents to solution withoutthe electrolytic processing step, a “mock” APC solution was generated.The “mock” APC solution contained the oxidant species that have beenmeasured in APC solutions: 0.6 mg/L free chlorine, 1 mg/L hydrogenperoxide, 9 μM nitrite, and 6.5 μM nitrate. Additionally, the saline andpH levels were the same as the APC solution administered to cells. Notethat due to the transient nature of ozone, ozone was not included in the“mock” APC solution. FIG. 5 shows graphs of human pancreatic cancer cellproliferation for APC solution and a “mock” APC solution, for thefollowing dosages: 1, 5, 12.5, 25, and 50 v/v %, illustrating that the“mock” APC solution does not yield the same dose-dependent pancreaticcancer cell growth inhibition as does the APC solution. Thus, thetherapeutic effects of APC solutions are dependent on theplasma-specific species generated.

EXAMPLE 5

EXAMPLE 5 illustrates how the plasma residence time will impact theoxidant species generated in the final APC solution, thus directlyimpacting its therapeutic efficacy. Employing an established oxidantanalysis method, the total oxidant levels were measured as a function ofresidence time of ultra-pure water as exposed to plasma. The totaloxidant levels were quantified using a derivatization assay employedwhereby triphenylphosphine (TPP) is oxidized to TPPO in the presence ofoxidant species, where the TPPO product is detected via HPLC-UV(Pinkernell et al., Anal. Chem. 69:3623-3627, 1997). FIG. 6 is a graphof the oxidant level as a function of residence time in the plasma,illustrating that the oxidant level increases as a function ofincreasing residence time in the reactor.

In many other experiments employing the plasma system described, theextent of oxidation of a surrogate molecule is directly related to thetreatment time. More specifically, since the residence time of thesolution in the plasma reactor is dependent upon both the solution flowrate and the average volume of solution in the reactor, decreasing theflow rate for a fixed volume will increase the residence time andvice-versa. Consistently, over a variety of different parameters, whenthe flow rate was doubled from 3 gph to 6 gph, there was a decrease inthe ability of the effluent solution to chemically modify surrogatemolecules. For example, utilizing the THDPR generating plasma underconditions in which the submersed pin electrodes are rotating at ratescapable of inducing turbulent flow conditions within the reactor with anelectrode distance of 1.5 mm, defined as the distance between therotating pin electrodes and stationary outer electrode, an input currentof 3 A, a reactor volume of 400 mL, and a flow rate of 3 gph(corresponding to a 2 min. residence time), a 15% reduction in theconcentration of methylene blue was observed, indicating a chemicalmodification of the surrogate molecule. When the flow rate was increasedto 6 gph (1 min. residence time), less than a 5% decrease in methyleneblue concentration was observed due to an overall decrease in reactivityof the chemical intermediate species formed for the lower-residence-timetrials where the water was in contact with the plasma/electrochemicalprocess for a shorter period of time.

EXAMPLE 6

The oxidation-reduction potential (ORP) is a common measure of aqueoussolution chemical species reactivity. In the above EXAMPLES, plasmadischarges were employed during the duration of the solution treatmenttime. EXAMPLE 6 describes the dependence of solution ORP on plasmadischarges during plasma on/off cycles, where no current was beingapplied during the off cycle. FIG. 7 is a graph of the measured ORP as afunction of time for a specific example of a 300 mg/L TDS influentsolution where the power was turned on and off to yield periods ofplasma discharge and no current flowing, respectively. When plasma wasdischarged, the ORP response increased, indicating an increase inaqueous chemotherapeutic species present in solution; subsequently, whenno current was introduced, the ORP value dropped, indicating thatchemical intermediates were not being formed or were formed in decreasedconcentrations.

EXAMPLE 7

EXAMPLE 7 illustrates how the applied current/power impacts theoxidizing capacity of the species formed. The THDPR can be run undercurrent or power control mode. In either case, an increase in the poweror current applied to the system has been found to yield a linearincrease in the oxidizing capacity of the system, as measured bychromophore loss (oxidation) of a model organic compound, Yellow 5. Morespecifically, FIG. 8 demonstrates how increasing the applied currentfrom 0.25 A to 1 A yields a linear increase in oxidation of Yellow 5.Similarly, FIG. 9 shows how increasing the applied power from 50 to 500W also yields a linear increase in the oxidation of Yellow 5.

EXAMPLE 8

EXAMPLE 8 illustrates how the spin rate of the THDPR as related tosystem turbulence, impacts the oxidizing capacity of the system relatedto the species formed. Using the THDPR, which contains a centralrotating array of negatively charged electrodes, the spin rate of therotor was found to impact the oxidizing capacity of the system, asindicated by the oxidation of a surrogate organic compound, MethyleneBlue, as measure spectroscopically. For influent solutions with a totaldissolved solids level of 1000 mg/L, higher spin rates (1000 rpm) werefound to result in ˜20% greater organics oxidation compared to lowerspin rates (700 rpm) for a given set of conditions. Thus, the spin rateof the rotor relating to the turbulence in the system may alter thedistribution of the generated species in solution.

EXAMPLE 9

EXAMPLE 9 illustrates how the TDS of the influent solution impacts theoxidizing capacity of the system related to the species formed. For theTHDPR generating plasma under conditions in which the submersed pinelectrodes are rotating at rates capable of inducing turbulent flowconditions within the reactor while applying an input current of 3 A andan aqueous solution residence time of approximately 2 min., a 2.5 mg/LTDS influent solution concentration resulted in the generation of 1 ppmhydrogen peroxide and a resultant effluent ORP value of 300 mV. When theTDS of the influent was increased to 1000 mg/L using NaCl, a 4 ppmconcentration of hydrogen peroxide was generated with a final ORP of 560mV, indicating that, under these operating conditions, an increase inTDS resulted in a subsequent increase in the chemical species formed insolution. Additionally, for these trials a surrogate organic compound,methylene blue, was added at 6 ppm into the influent solution and theresultant decrease in methylene blue concentration was measured viaabsorbance spectrophotometry as the species underwent reaction with thechemical species formed during operation of the plasma reactor. For the2.5 mg/L TDS influent solution, a 15% reduction in methylene blueconcentration was found, whereas the 1000 mg/L TDS solution yielded a35% decrease in methylene blue. Since a variety of species thatcontribute to ORP with the potential to modify organic compounds alsoare responsible for the reaction of methylene blue, a larger percentdecrease in methylene blue concentration indicates that greater chemicalreactivity was observed in the higher TDS solution during plasmatreatment.

EXAMPLE 10

EXAMPLE 10 illustrates how the ability to generate plasma can bedependent upon both solution conductivity and electrode gap. For a givenset of conditions, as shown in FIG. 10 , an electrode separationdistance of 1 mm restricted generation of plasma to influent solutionswith less than about 5,000 mg/L TDS, while increasing the separationdistance to 1.5 mm enabled plasma generation in influent solutions with0-15,000 mg/L TDS. Further increasing the distance to 2 mm resulted inmaintained plasma operation at greater than 15,000 mg/L, but would notenable plasma generation at <3 mg/L TDS. These data, when consideredtogether, suggest that the ability to generate plasmas for a givenelectrode separation distance is dependent upon the influent TDS, wheresmaller separation distances enable plasma generation at zero-to-low TDSsamples and an increase in separation enables plasma generation athigher TDS. Since variable ion concentrations due to the TDS level willimpact the lifetime of intermediate species, the plasma processes thatoccur for different TDS solutions will impact the overall nature of thereactive species present in the final EPAAC solution. For example,reactive intermediates such as ozone are decomposed in the presence ofchloride ion; thus, increasing chloride concentrations can decrease thelifetime of ozone in solution, which impacts the overall nature of theEPAAC solution and its ability to impact cellular processes.

EXAMPLE 11

EXAMPLE 11 illustrates how the gas flow rate impacts the oxidizingcapacity of the system related to the species formed. Using the THDPRconfiguration, the flow rate of house air into the reactor, where theair flow results in microbubbles forming at the tip of the electrode,dictates the resulting oxidizing capacity of the system, as measured bythe chromophore loss of the organic compound, Yellow 5. When increasingthe gas flow rate from 50 to 100 SCFH, an increase in the Yellow 5chromophore loss was increased from 2 to 12.5%, respectively, under thesame set of reaction conditions. This represents a linear region between50 and 100 SCFH air flow rate where an increase in flow rate resulted inmore oxidation. However, further increasing the air flow rate up to 200SCFH did not yield any significant increase in oxidation compared to 100SCFH. Thus, the gas flow rate may impact the oxidizing capacity of thesystem.

EXAMPLE 12

EXAMPLE 12 illustrates the prolonged shelf-life of the APC solution. Forcommercial applications, the shelf-life of the APC solution product willdictate its mode of distribution into the market. Preliminary shelf-liferesults have indicated that APC solutions maintain the same physicalproperties as measured by oxidation-reduction potential, pH, TDS, andfree chlorine, for solutions stored under refrigeration for 4 d,suggesting that 4 d is a lower limit for the shelf-life of thesesolutions. This is indirect evidence that the EPAAC solutions retaintheir chemotherapeutic activity for this period as well.

The foregoing description of the invention has been presented forpurposes of illustration and description and is not intended to beexhaustive or to limit the invention to the precise form disclosed, andobviously many modifications and variations are possible in light of theabove teaching. The embodiments were chosen and described in order tobest explain the principles of the invention and its practicalapplication to thereby enable others skilled in the art to best utilizethe invention in various embodiments and with various modifications asare suited to the particular use contemplated. It is intended that thescope of the invention be defined by the claims appended hereto.

What is claimed is:
 1. A method for treating pancreatic cancer, comprising: providing a solution having a chosen quantity of a single sodium chloride solute dissolved in water; circulating said single sodium chloride solute solution through an atmospheric pressure air plasma discharge generated using a high-density plasma reactor for a selected period of time, forming thereby a plasma activated solution having a pH; adding a base to said plasma activated solution to adjust the pH to 7.4±0.1, forming thereby a physiological solution; adding sodium chloride to said physiological solution to adjust the chosen quantity of sodium chloride to 9±0.1 g per liter of solution, forming thereby an isotonic solution; and and exposing cells afflicted with pancreatic cancer to said isotonic solution.
 2. The method of claim 1, wherein said step of exposing cells afflicted with pancreatic cancer to said isotonic solution further comprises the step of injecting said isotonic solution into a pancreas.
 3. The method of claim 1, wherein said step of exposing cells afflicted with pancreatic cancer to said isotonic solution further comprises the step of injecting said isotonic solution into tissue surrounding a pancreas.
 4. The method of claim 1, wherein said step of exposing cells afflicted with pancreatic cancer to said isotonic solution further comprises the step of intravenously injecting said isotonic solution into a patient.
 5. The method of claim 1, wherein treating pancreatic cancer comprises reducing proliferation of cells afflicted with pancreatic cancer.
 6. The method of claim 5, wherein proliferation of normal cells is unaffected by said treatment of pancreatic cancer.
 7. The method of claim 1, wherein the chosen quantity of said single sodium chloride solute dissolved in water is 1 g/L. 